Summary of work: This project focuses on signal transduction pathways mediating the molecular reponse to stress in normal and aged cells. Studies over the past year have concentrated on three topics. (1) Identification of upstream mediators in arsenite-triggered ERK cascade. Previously we demonstrated that arsenite can activate ERK, JNK and p38 MAP kinases. Recent studies have focused on the role of growth factor receptors in mediating ERK activation. We have shown that arsenite treatment results in the rapid activation of epidermal growth factor receptor (EGFR), tyrosine phosphorylation of the Shc adaptor, and the formation of EGFR-Shc-Grb2 complexes. These events, as well as activation of ERK, were all drastically reduced by down-regulation of EGFR activity. These results provide the first evidence that the EGFR and Shc are critical mediators in the activation of the Ras/ERK signaling cascade by arsenite and suggest that this tumor promoter acts largely by usurping this growth factor signaling pathway. (2) Structural basis of MAP kinase phosphatase-1(MKP-1). MAP kinase phosphatases are a group of dual specificity phosphatases induced by extracellular stimuli including growth factors and stress. They can exhibit selectivity towards different members of the MAP kinase family. The goal of this study is to understand the structural basis for MKP-1 substrate specificity. Various domains of MKP-1 are being swapped with corresponding regions of PAC-1 or MKP-3 to generate chimeras. Analyzing the binding and enzymatic specificities of these chimeras for different MAP kinases may provide important information about the structural basis for the substrate selectivity. (3) Age-associated alteration in signaling pathways in rat hepatocytes. Previously we demonstrated that aging is correlated with decreases in both ERK MAP kinase and p70 S6 kinase activities following growth factor treatment. A decline in the activities of both kinases suggests that aged cells may display an alteration in an early upstream event common to these pathways. We have compared the earliest signaling events which occur in response to EGF stimulation in young and aged cells. In young hepatocytes, EGF triggers rapid tyrosine phosphorylation of EGFR and Shc, and complex formation between them. Formation of this complex in response to EGF is significantly reduced in aged cells, although no difference in tyrosine-phosphorylation of either EGFR or Shc is observed. The alteration in the growth factor receptor complexes may contribute to the decline in proliferation capacity in aged cells.